Methods for assaying proteins in aqueous liquids such as biological liquids (blood, serum and urine) are known. Both wet and dry methods are known. Such assays are carried out for a variety of reasons. For example, the knowledge of protein levels in human serum and urine is important in the diagnosis of such conditions as viral or bacterial meningitis.
Wet methods refer to those methods in which the clinical reagents are first dissolved or suspended in a liquid aqueous vehicle. Dry chemical methods have reference to chemical methods which are performed using reagent composition incorporated in various substantially "dry-to-the-touch" elements. Examples of such elements include "dip and read" test strips and multi-zone analytical test elements. The latter elements are disclosed for example, in U.S. Pat. No. 4,132,528.
The chemical reaction used extensively in both wet and dry protein assay methods is the "biuret" reaction. The general reaction is described in "Determination of Serum Protein by Means of the Biuret Reaction" prepared by A. G. Gornall et al appeared in Journal of Biology Chemistry, Vol. 177, page 751 (1949).
In general the biuret reaction involves a "biuret" reagent which contains the chelated cupric form of copper and a base composition of sufficient strength to provide a pH in excess of about 12). When protein in an aqueous liquid such as serum interacts with the biuret reagent, a reaction between the cupric form of copper and the protein occurs to produce a violet color. The color intensity is directly proportional to the protein content of the serum. Thus, the protein level can be measured by well known colorimetric analytical techniques.
In the element of U.S. Pat. No. 4,132,528 the analytical element is a multi-zone analytical element. The element comprises a spreading zone or layer in fluid contact with a reagent zone or layer. The reagent layer or zone is coated on a support such as polyethylene terephthalate. The biuret composition is included in the reagent zone. The reagent zone also includes an alkaline producing composition of sufficient concentration to maintain a pH in excess of 12. This element, while extremely useful and convenient, has the disadvantage that it has low sensitivity in that it cannot detect protein level at 100 mg/dl or below.